5,345 research outputs found
Novel 129Xe Magnetic Resonance Imaging and Spectroscopy Measurements of Pulmonary Gas-Exchange
Gas-exchange is the primary function of the lungs and involves removing carbon dioxide from the body and exchanging it within the alveoli for inhaled oxygen. Several different pulmonary, cardiac and cardiovascular abnormalities have negative effects on pulmonary gas-exchange. Unfortunately, clinical tests do not always pinpoint the problem; sensitive and specific measurements are needed to probe the individual components participating in gas-exchange for a better understanding of pathophysiology, disease progression and response to therapy.
In vivo Xenon-129 gas-exchange magnetic resonance imaging (129Xe gas-exchange MRI) has the potential to overcome these challenges. When participants inhale hyperpolarized 129Xe gas, it has different MR spectral properties as a gas, as it diffuses through the alveolar membrane and as it binds to red-blood-cells. 129Xe MR spectroscopy and imaging provides a way to tease out the different anatomic components of gas-exchange simultaneously and provides spatial information about where abnormalities may occur.
In this thesis, I developed and applied 129Xe MR spectroscopy and imaging to measure gas-exchange in the lungs alongside other clinical and imaging measurements. I measured 129Xe gas-exchange in asymptomatic congenital heart disease and in prospective, controlled studies of long-COVID. I also developed mathematical tools to model 129Xe MR signals during acquisition and reconstruction. The insights gained from my work underscore the potential for 129Xe gas-exchange MRI biomarkers towards a better understanding of cardiopulmonary disease. My work also provides a way to generate a deeper imaging and physiologic understanding of gas-exchange in vivo in healthy participants and patients with chronic lung and heart disease
Engineering Systems of Anti-Repressors for Next-Generation Transcriptional Programming
The ability to control gene expression in more precise, complex, and robust ways is becoming increasingly relevant in biotechnology and medicine. Synthetic biology has sought to accomplish such higher-order gene regulation through the engineering of synthetic gene circuits, whereby a geneâs expression can be controlled via environmental, temporal, or cellular cues. A typical approach to gene regulation is through transcriptional control, using allosteric transcription factors (TFs). TFs are regulatory proteins that interact with operator DNA elements located in proximity to gene promoters to either compromise or activate transcription. For many TFs, including the ones discussed here, this interaction is modulated by binding to a small molecule ligand for which the TF evolved natural specificity and a related metabolism. This modulation can occur with two main phenotypes: a TF shows the repressor (X+) phenotype if its binding to the ligand causes it to dissociate from the DNA, allowing transcription, while a TF shows the anti-repressor (XA) phenotype if its binding to the ligand causes it to associate to the DNA, preventing transcription. While both functional phenotypes are vital components of regulatory gene networks, anti-repressors are quite rare in nature compared to repressors and thus must be engineered.
We first developed a generalized workflow for engineering systems of anti-repressors from bacterial TFs in a family of transcription factors related to the ubiquitous lactose repressor (LacI), the LacI/GalR family. Using this workflow, which is based on a re-routing of the TFâs allosteric network, we engineered anti-repressors in the fructose repressor (anti-FruR â responsive to fructose-1,6-phosphate) and ribose repressor (anti-RbsR â responsive to D-ribose) scaffolds, to complement XA TFs engineered previously in the LacI scaffold (anti-LacI â responsive to IPTG). Engineered TFs were then conferred with alternate DNA binding. To demonstrate their utility in synthetic gene circuits, systems of engineered TFs were then deployed to construct transcriptional programs, achieving all of the NOT-oriented Boolean logical operations â NOT, NOR, NAND, and XNOR â in addition to BUFFER and AND. Notably, our gene circuits built using anti-repressors are far simpler in design and, therefore, exert decreased burden on the chassis cells compared to the state-of-the-art as anti-repressors represent compressed logical operations (gates).
Further, we extended this workflow to engineer ligand specificity in addition to regulatory phenotype. Performing the engineering workflow with a fourth member of the LacI/GalR family, the galactose isorepressor (GalS â naturally responsive to D-fucose), we engineered IPTG-responsive repressor and anti-repressor GalS mutants in addition to a D-fucose responsive anti-GalS TF. These engineered TFs were then used to create BANDPASS and BANDSTOP biological signal processing filters, themselves compressed compared to the state-of-the-art, and open-loop control systems. These provided facile methods for dynamic turning âONâ and âOFFâ of genes in continuous growth in real time. This presents a general advance in gene regulation, moving beyond simple inducible promoters.
We then demonstrated the capabilities of our engineered TFs to function in combinatorial logic using a layered logic approach, which currently stands as the state-of-the art. Using our anti-repressors in layered logic had the advantage of reducing cellular metabolic burden, as we were able to create the fundamental NOT/NOR operations with fewer genetic parts. Additionally, we created more TFs to use in layered logic approaches to prevent cellular cross-talk and minimize the number of TFs necessary to create these gene circuits. Here we demonstrated the successful deployment of our XA-built NOR gate system to create the BUFFER, NOT, NOR, OR, AND, and NAND gates.
The work presented here describes a workflow for engineering (i) allosteric phenotype, (ii) ligand selectivity, and (iii) DNA specificity in allosteric transcription factors. The products of the workflow themselves serve as vital tools for the construction of next-generation synthetic gene circuits and genetic regulatory devices. Further, from the products of the workflow presented here, certain design heuristics can be gleaned, which should better facilitate the design of allosteric TFs in the future, moving toward a semi-rational engineering approach. Additionally, the work presented here outlines a transcriptional programming structure and metrology which can be broadly adapted and scaled for future applications and expansion. Consequently, this thesis presents a means for advanced control of gene expression, with promise to have long-reaching implications in the future.Ph.D
Rational development of stabilized cyclic disulfide redox probes and bioreductive prodrugs to target dithiol oxidoreductases
Countless biological processes allow cells to develop, survive, and proliferate. Among these, tightly balanced regulatory enzymatic pathways that can respond rapidly to external impacts maintain dynamic physiological homeostasis. More specifically, redox homeostasis broadly affects cellular metabolism and proliferation, with major contributions by thiol/disulfide oxidoreductase systems, in particular, the Thioredoxin Reductase Thioredoxin (TrxR/Trx) and the Glutathione Reductase-Glutathione-Glutaredoxin (GR/GSH/Grx) systems.
These cascades drive vital cellular functions in many ways through signaling, regulating other proteins' activity by redox switches, and by stoichiometric reductant transfers in metabolism and antioxidant systems. Increasing evidence argues that there is a persistent alteration of the redox environment in certain pathological states, such as cancer, that heavily involve the Trx system: upregulation and/or overactivity of the Trx system may support or drive cancer progression, making both TrxR and Trx promising targets for anti-cancer drug development.
Understanding the biochemical mechanisms and connections between certain redox cascades requires research tools that interact with them. The state-of-the-art genetic tools are mostly ratiometric reporters that measure reduced:oxidized ratios of selected redox pairs or the general thiol pool. However, the precise cellular roles of the central oxidoreductase systems, including TrxR and Trx, remain inaccessible due to the lack of probes to selectively measure turnover by either of these proteins. However, such probes would allow measuring their effective reductive activity apart from expression levels in native systems, including in cells, animals, or patient samples. They are also of high interest to identify chemical inhibitors for TrxR/Trx in cells and to validate their potential use as anti-cancer agents (to date, there is no selective cellular Trx inhibitor, and most known TrxR inhibitors were not comprehensively evaluated considering selectivity and potential off-targets). However, small molecule redox imaging tools are underdeveloped: their protein specificity, spectral properties, and applicability remain poorly precedented.
This work aimed to address this opportunity gap and develop novel, small molecule diagnostic and therapeutic tools to selectively target the Trx system based on a modular trigger cargo design: artificial cyclic disulfide substrates (trigger) for oxidoreductases are tethered to molecular agents (cargo) such that the cargoâs activity is masked and is re-established only through reduction by a target protein.
The rational design of these novel reduction sensors to target the cell's strongest disulfide-reducing enzymes was driven by the following principles: (i) cyclic disulfide triggers with stabilized ring systems were used to gain low reduction potentials that should resist reduction except by the strongest cellular reductases, such as Trx; and (ii) the cyclic topology also offers the potential for kinetic reversibility that should select for dithiol-type redox proteins over the cellular monothiol background. Creating imaging agents based on such two-component designs to selectively measure redox protein activity in native cells required to combine the correct trigger reducibility, probe activation kinetics, and imaging modalities and to consider the overall molecular architecture.
The major prior art in this field has applied cyclic 5-membered disulfides (1,2 dithiolanes) as substrates for TrxR in a similar way to create such tools. However, this motif was described elsewhere as thermodynamically instable and was due to widely used for dynamic covalent cascade reactions. By comparing a novel 1,2 dithiolane-based probe to the state-of-the-art probes, including commercial TrxR sensors, by screening a conclusive assay panel of cellular TrxR modulations, I clarified that 1,2 dithiolanes are not selective substrates for TrxR in biological settings (Nat Commun 2022).
Instead, aiming for more stable ring systems and thus more robust redox probes, during this work, I developed bicyclic 6 membered disulfides (piperidine fused 1,2 dithianes) with remarkably low reduction potentials. I showed that molecular probes using them as reduction sensors can be mostly processed by thioredoxins while being stable against reduction by GSH. The thermodynamically stabilized decalin like topology of the cis-annelated 1,2 dithianes requires particularly strong reductants to be cleaved. They also select for dithiol type redox proteins, like Trx, based on kinetic reversibility and offer fast cyclization due to the preorganization by annelation (JACS 2021).
This work further expanded the systemâs modularity with structural cores based on piperazine-fused 1,2 dithianes with the two amines allowing independent derivatization. Diagnostic tools using them as reduction sensors proved equally robust but with highly improved activation kinetics and were thus cellularly activated. Cellular studies evolved that they are substrates for both Trxs and their protein cousins Grxs, so measuring the cellular dithiol protein pool rather than solely Trx activity (preprint 2023).
Finally, a trigger based on a slightly adapted reduction sensor, a desymmetrized 1,2 thiaselenane, was designed for selective reduction by TrxRâs selenol/thiol active site, then combined with a precipitating large Stokesâ shift fluorophore and a solubilizing group, to evolve the first selective probe RX1 to measure cellular TrxR activity, which even allowed high throughput inhibitor screening (Chem 2022).
The central principle of this work was further advanced to therapeutic prodrugs based on the duocarmycin cargo (CBI) with tunable potency (JACS Au 2022) that can be used to create off-to-on therapeutic prodrugs. Such CBI prodrugs employing stabilized 1,2 dichalcogenide triggers proved to be cytotoxins that depend on Trx system activity in cells. They could further be exploited for cell-line dependent reductase activity profiling by screening their redox activation indices, the reduction-dependent part of total prodrug activation, in 177 cell lines. Beyond that, these prodrugs were well-tolerated in animals and showed anti-cancer efficacy in vivo in two distinct mouse tumor models (preprint 2022).
Taken together, I introduced unique monothiol-resistant reducible motifs to target the cellular Trx system with chemocompatible units for each for TrxR and Trx/Grx, where the cyclic nature of the dichalcogenides avoids activation by GSH. By using them with distinct molecular cargos, I developed novel selective fluorescent reporter probes; and introduced a new class of bioreductive therapeutic constructs based on a common modular design. These were either applied to selectively measure cellular reductase activity or to deliver cytotoxic anti cancer agents in vivo. Ongoing work aims to differentiate between the two major redox effector proteins Trx and Grx, requiring additional layers of selectivity that may be addressed by tuned molecular recognition. The flexible use of various molecular cargos allows harnessing the same cellular redox machinery by either probes or prodrugs. This allows predictive conclusions from diagnostics to be directly translated into therapy and offers great potential for future adaptation to other enzyme classes and therapeutic venues.Die zellulĂ€re Redox-Homöostase hĂ€ngt von Thiol/Disulfid-Oxidoreduktasen ab, die den Stoffwechsel, die Proliferation und die antioxidative Antwort von Zellen beeinflussen. Die wichtigsten Netzwerke sind die Thioredoxin Reduktase-Thioredoxin (TrxR/Trx) und Glutathion Reduktase-Glutathion-Glutaredoxin (GR/GSH/Grx) Systeme, die ĂŒber Redox-Schalter in Substratproteinen lebenswichtige zellulĂ€re Funktionen steuern und so an der Redox-Regulation und -SignalĂŒbertragung beteiligt sind. Persistente VerĂ€nderungen des Redoxmilieus in pathologischen ZustĂ€nden, wie z. B. bei Krebs, sind in hohem MaĂe mit dem Trx-System verbunden. Eine Hochregulierung und/oder ĂberaktivitĂ€t des Trx-Systems, die bei vielen Krebsarten auftreten, unterstĂŒtzt zudem das Fortschreiten des Krebswachstums, was TrxR/Trx zu vielversprechenden Zielproteinen fĂŒr die Entwicklung neuer Krebsmedikamente macht.
Um die biochemischen Prozesse dahinter zu erforschen, sind spezielle Techniken zur Visualisierung und Messung enzymatischer AktivitĂ€t nötig. Die hierzu geeigneten, meist genetischen Sensoren messen ratiometrisch das VerhĂ€ltnis reduzierter/oxidierter Spezies in zellulĂ€rem Umfeld oder spezifisch ausgewĂ€hlte Redoxpaare. Die weitere Erforschung der exakten Funktion von TrxR/Trx und deren Substrate ist jedoch durch mangelnde Nachweismethoden limitiert. Diese sind auĂerdem zur Validierung chemischer Hemmstoffe fĂŒr TrxR/Trx in Zellen und deren potenziellen Verwendung als Krebsmittel von groĂem Interesse. Bislang gibt es keinen selektiven zellulĂ€ren Trx-Inhibitor und potenzielle Off-Target-Effekte der bekannten TrxR-Inhibitoren wurden nicht abschlieĂend bewertet.
Ziel dieser Arbeit ist die Entwicklung niedermolekularer, diagnostischer und therapeutischer Werkzeuge, die selektiv auf das Trx-System abzielen und auf einem modularen Trigger-Cargo Design basieren. Hierzu werden zyklische Disulfid-Substrate (Trigger) fĂŒr Oxidoreduktasen so mit molekularen Wirkstoffen (Cargo) verknĂŒpft, dass dabei die WirkstoffaktivitĂ€t maskiert, und erst nach Reduktion durch ein Zielprotein wiederhergestellt wird. Diese neuartigen, synthetischen Reduktionssensoren basieren auf den folgenden Grundprinzipien: (i) Zyklische Disulfide sind thermodynamisch stabilisiert und können nur durch die stĂ€rksten Reduktasen gespalten werden; und (ii) die zyklische Topologie ermöglicht die kinetische ReversibilitĂ€t der zwei Thiol-Disulfid-Austauschreaktionen, die eine erste Reaktion mit Monothiolen, wie z. B. GSH, sofort umkehrt und so eine vollstĂ€ndige Reduktion verhindert.
Die meisten frĂŒheren Arbeiten auf diesem Gebiet verwendeten ein zyklisches, fĂŒnfgliedriges Disulfid (1,2 Dithiolan) als Substrat fĂŒr TrxR. Das gleiche Strukturmotiv wurde jedoch an anderer Stelle als thermodynamisch instabil beschrieben und aufgrund dieser Eigenschaft explizit fĂŒr dynamische Kaskadenreaktionen verwendet. Deshalb vergleicht diese Arbeit zu Beginn einen neuen 1,2 Dithiolan basierten fluorogenen Indikator mit bestehenden, z. T. kommerziellen, Redox Sonden fĂŒr TrxR in einer Reihe von Zellkultur-Experimenten unter Modulation der zellulĂ€ren TrxR AktivitĂ€t und stellt so einen Widerspruch in der Literatur klar: 1,2 Dithiolane eignen sich nicht als selektive Substrate fĂŒr TrxR, da sie labil sowohl gegen die Reduktion durch andere Redoxproteine, als auch gegen den Monothiol Hintergrund in Zellen sind (Nat. Commun. 2022).
Als alternatives Strukturmotiv wird in dieser Arbeit ein bizyklisches sechsgliedriges Disulfid (anneliertes 1,2 Dithian) etabliert. Durch sein niedriges Reduktionspotenzial, also seine hohe Resistenz gegen Reduktion, werden molekulare Sonden basierend auf diesem 1,2 Dithian als Reduktionssensor fast ausschlieĂlich von Trx aktiviert, nicht aber von TrxR oder GSH (JACS 2021). Dieses Kernmotiv bestimmt dabei die Reduzierbarkeit, und damit die EnzymspezifitĂ€t, durch seine zyklische Natur und die Annelierung, auch unter Verwendung unterschiedlicher Farb-/Wirkstoffe. Auf dieser Grundlage konnte die molekulare Struktur durch einen weiteren Modifikationspunkt fĂŒr die flexible Verwendung weiterer funktioneller Einheiten ergĂ€nzt werden. Obwohl zellulĂ€re Studien ergaben, dass diese neuartigen 1,2 Dithian Einheiten in Zellen sowohl Trx als auch das strukturell verwandte Grx adressieren, sind die daraus resultierenden diagnostischen MolekĂŒle wertvoll, um den katalytischen Umsatz zellulĂ€rer Dithiol-Reduktasen, der sogenannten Trx Superfamilie, selektiv anzuzeigen (Preprint 2023).
BegĂŒnstigt durch das modulare MolekĂŒldesign stellt diese Arbeit zudem das erste Reportersystem RX1 zum selektiven Nachweis der TrxR-AktivitĂ€t in Zellen vor. Es basiert auf der Verwendung eines zyklischen, unsymmetrischen Selenenylsulfid-Sensors (1,2 Thiaselenan), der selektiv von dem einzigartigen Selenolat der TrxR angegriffen wird, und dadurch letztlich nur von TrxR reduziert werden kann. RX1 eignete sich zudem fĂŒr eine Hochdurchsatz-Validierung bestehender TrxR Inhibitoren und unterstreicht dadurch den kommerziellen Nutzen derartiger Diagnostika (Chem 2022).
Das zentrale Trigger-Cargo Konzept dieser Arbeit wurde fĂŒr therapeutische Zwecke weiterentwickelt und nutzt dabei den einzigartigen Wirkmechanismus der Duocarmycin-Naturstoffklasse (CBI) (JACS Au 2022) zur Entwicklung reduktiv aktivierbarer Therapeutika. CBI Prodrugs basierend auf stabilisierten Redox-Schaltern (1,2 Dithiane fĂŒr Trx; 1,2 Thiaselenan fĂŒr TrxR) reagierten signifikant auf TrxR-Modulation in Zellen. Sie wurden darĂŒber hinaus durch das Referenzieren ihrer AktivitĂ€t gegenĂŒber nicht-reduzierbaren KontrollmolekĂŒle fĂŒr die Erstellung zelllinienabhĂ€ngiger Profile der ReduktaseaktivitĂ€t in 177 Zelllinien genutzt. SchlieĂlich waren diese neuen Krebsmittel im Tiermodell gut vertrĂ€glich und zeigten in zwei verschiedenen Mausmodellen eine krebshemmende Wirkung (Preprint 2022b).
Zusammenfassend prĂ€sentiert diese Dissertation monothiol-resistente reduzierbare Trigger-Einheiten fĂŒr das zellulĂ€re Trx-System zur Entwicklung neuartiger, selektiver Reporter-Sonden, sowie eine neue Klasse reduktiv aktivierbarer Krebsmittel auf Basis eines adaptierbaren Trigger-Cargo Designs. Diese fanden entweder zur selektiven Messung zellulĂ€rer ProteinaktivitĂ€t oder zum Einsatz als Antikrebsmittel Verwendung. Es wurden chemokompatible Motive sowohl fĂŒr TrxR als auch fĂŒr Trx/Grx identifiziert, wobei deren zyklische Natur eine Aktivierung durch GSH verhindert. Eine weitere Differenzierung zwischen den beiden Redox-Proteinen Trx und Grx und anderen Proteinen der Trx-Superfamilie erfordert eine zusĂ€tzliche Ebene der Selektierung, z. B. durch molekulare Erkennung, und ist Gegenstand laufender Arbeiten.
Die flexible Verwendung verschiedener molekularer Wirkstoffe ermöglicht dabei die âPipeline-Entwicklungâ von Diagnostika und Therapeutika, die von der zellulĂ€ren Redox-Maschinerie analog umgesetzt werden, und dadurch Schlussfolgerungen aus der Diagnostik direkt auf eine Therapie ĂŒbertragbar machen. Dies birgt groĂes Potenzial fĂŒr kĂŒnftige Entwicklungen bei einer potenziellen Ăbertragung des modularen Konzepts auf andere Enzymklassen und therapeutische Einsatzgebiete
Synthetic Aperture Radar (SAR) Meets Deep Learning
This reprint focuses on the application of the combination of synthetic aperture radars and depth learning technology. It aims to further promote the development of SAR image intelligent interpretation technology. A synthetic aperture radar (SAR) is an important active microwave imaging sensor, whose all-day and all-weather working capacity give it an important place in the remote sensing community. Since the United States launched the first SAR satellite, SAR has received much attention in the remote sensing community, e.g., in geological exploration, topographic mapping, disaster forecast, and traffic monitoring. It is valuable and meaningful, therefore, to study SAR-based remote sensing applications. In recent years, deep learning represented by convolution neural networks has promoted significant progress in the computer vision community, e.g., in face recognition, the driverless field and Internet of things (IoT). Deep learning can enable computational models with multiple processing layers to learn data representations with multiple-level abstractions. This can greatly improve the performance of various applications. This reprint provides a platform for researchers to handle the above significant challenges and present their innovative and cutting-edge research results when applying deep learning to SAR in various manuscript types, e.g., articles, letters, reviews and technical reports
Electroencephalography-Based BrainâMachine Interfaces in Older Adults: A Literature Review
The aging process is a multifaceted phenomenon that affects cognitive-affective and physical functioning as well as interactions with the environment. Although subjective cognitive decline may be part of normal aging, negative changes objectified as cognitive impairment are present in neurocognitive disorders and functional abilities are most impaired in patients with dementia. Electroencephalography-based brainâmachine interfaces (BMI) are being used to assist older people in their daily activities and to improve their quality of life with neuro-rehabilitative applications. This paper provides an overview of BMI used to assist older adults. Both technical issues (detection of signals, extraction of features, classification) and application-related aspects with respect to the usersâ needs are considered
- âŠ